| Interferon(IFN)is a cytokine induced by the body during viral infection and it is an important role in innate immunity.It can be divided into three categories,including type I interferon,type II interferon,and type III interferon.Type III interferon(IFN-λs)mainly plays an important role on the surface of host mucosal cells in three of them.At present,IFNλs and its subtypes have been found in many species such as pigs,monkeys,and rats,but chicken IFNλs(ch IFN-λ)has been reported less,and only ch IFN-λ3 has been found.In the early stage of the research of ch IFN-λ,a new chicken interferon type 3 spliceosome ch IFN-λ3a was discovered.In this study,the activity was verified by transient overexpression,and its antiviral activity was studied by baculovirus/insect cell expression system for expression and purification,in order to lay a solid foundation for the mechanism and clinical study of ch IFN-λ3a.The specific methods and results are as follows:(1)ch IFN-λ3a plasmid construction,expression and analysis of antiviral activityBased on the ch IFN-λ3a obtained earlier,it was subcloned into the eukaryotic expression vector pc DNA-3.1 to obtain the recombinant plasmid pc DNA3.1-ch IFN-λ3a,and pc DNA3.1-ch IFN-λ3 was constructed by the same method as a control.After the digestion and sequencing were correct,the plasmid was transfected into DF-1 cells by liposome transfection for expression verification,and the Western blot assay showed that ch IFN-λ3a and ch IFN-λ3were successfully expressed.On this basis,the inhibitory effect of ch IFN-λ3a on VSV-GFP virus was analyzed by viral infection experiment,and the results showed that transient expression of ch IFN-λ3a and ch IFN-λ3 could inhibit the proliferation of VSV-GFP virus in DF-1 cells.(2)ch IFN-λ3a expression and identification in recombinant baculovirusesIn order to explore the expression capacity of different baculovirus vectors,three vectors,p Fast Bac1,p Fast Bac-Dual,and p Fast Bac-DN(modified for this laboratory)were selected.After adding Strep tags at the 3’ end of ch IFN-λ3a gene,insect cell codons were optimized and synthesized,and three recombinant shuttle plasmids were obtained by double digestion and three vectors were linked,and after blue and white spot screening,the recombinant rod with successful transposition was selected,and after PCR verification was correct,transfected into SF-9 cells and the expression of the target protein was detected by Western blot.The results showed that all three vectors could successfully express the target protein,and p Fast Bac1 was the highest expression as the carrier target protein after screening with different virulence amounts and different time conditions,so the vector was determined as the best carrier for the expression of the target protein.(3)Purification and antiviral activity analysis of recombinant ch IFN-λ3aThe optimal vector determined in experiment(2)was used for large-scale expression of the protein of interest,and the Strep affinity chromatography column was used for purification to obtain ch IFN-λ3a with a purity of 42%.After the purified ch IFN-λ3a was measured by interferon activity,DF-1 cells were incubated,infected with VSV-GFP(Vesicular Stomatitis Virus-GFP)virus(0.01 MOI)after 12 h,and ch IFN-λ3a antiviral activity was analyzed by fluorescence observation,flow cytometry and crystal violet staining.At the same time,H9N2 subtype avian influenza virus(5 MOI)was inoculated for antiviral activity verification.The results showed that ch IFN-λ3a could inhibit the proliferation of VSV-GFP and H9N2 subtypes of avian influenza virus,indicating that ch IFN-λ3a was successfully expressed by recombinant baculovirus-insect system and had antiviral activity. |
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